Sperm recognize the mouse zona pellucida (ZP1, ZP2, ZP3) prior to, but not after, fertilization. Several biochemical changes have been inferred to account for this dichotomy, but only proteolytic cleavage of ZP2 has been experimentally demonstrated. We have proposed a model in which cleavage status of ZP2 determines whether the zona pellucida is permissive (intact ZP2) or non-permissive (cleaved ZP2) for sperm-egg recognition. To confirm the central role of ZP2 in sperm-egg recognition, the cleavage site was determined by microscale Edman degradation and a genomic clone of moZP2 was mutated at the cleavage site by DNA recombineering. Transgenic mouse lines with the mutant ZP2 (mutZP2) were established and each of six founder lines expressed both normal and mutated ZP2 proteins. Two lines were crossed into the Zp2 null background to generate mutZP2 rescue mice. Ovarian histology of 3-4 week old mutZP2 rescue females was unremarkable, although ovulated eggs from rescue mice had a thinner zona compared to normal eggs. MutZP2 rescue mice were fertile in vivo and sperm bound to and fertilized their eggs. However, as predicted by the model, sperm continued to adhere to the surface of the zona matrix surrounding 2-cell embryos despite fertilization and an effective block to polyspermy. The molecular basis by which ZP2 cleavage blocks sperm-egg recognitioin remains under investigation.